Toxigenic E.coli infection is one of the most common causes of childhood diarrhea in developing countries, where it is often accompanied by fatal dehydration, as well as the most common cause of traveler's diarrhea in adults. The heat-stable enterotoxin of E. coli (STa), a small cysteine- rich peptide, triggers fluid and electrolyte secretion by stimulating cyclic GMP. Molecular cloning studies by other investigators have shown that intestinal membrane-bound guanylate cyclase serves as a receptor for STa. Nevertheless, the domain(s) of the receptor important for toxin binding remain unknown. In addition, recent work by the author and collaborators has shown that the toxic actions of STa are enhanced by agents which activate protein kinase C (PKC), including phorbol esters and humoral agents able to activate PKC (carbachol, histamine). The interaction between PKC activators and STa can be observed in intact cells, broken cells, and in membranes treated with highly purified PKC and can be observed as an increase in cyclic GMP production and, in intact cell monolayers, as a marked synergistic chloride secretory response. The implications of these findings are that host susceptibility to the toxin may be modulated by neurohumoral signals in the host. The broad, long-term goals of this project are to understand the nature and function of the STa receptor/guanylate cyclase, and how the cGMP interacts with other signalling systems, especially PKC, in intestinal secretion. Specific aims include: (l) to determine the STa-binding domain or domains on the extracellular portion of intestinal gnanylate cyclase by using biochemical, immunologic, and site-directed mutagenic approaches; and (2) to determine whether the effects of PKC are, as we hypothesize, due to direct phosphorylation of guanylate cyclase, and if so to determine the site(s) of phosphorylation by phosphopeptide mapping and site-directed mutagenesis. The only modern therapeutic advance in the treatment of diarrhea, oral rehydration therapy, was introduced more than twenty years ago following the discovery of glucose-sodium cotransport in basic investigations. A rededication to understanding basic biochemical mechanisms controlling intestinal secretion and cell signalling may yield the clues needed for future advances in therapy which are so widely needed. Just as cholera toxin and pertussis toxin played key roles in elucidating the structure and function of G-proteins and adenylate cyclase the unusual, hormone-like toxin STa will likely lead to new understanding of intestinal guanylate cyclase and its regulation.